Structure determination of Methanocaldococcus jannaschii nucleoside kinase

Abstract

All organisms can be divided into the tree kingdoms of life: Archaea, Bacteria and Eukarya. Archaeal organisms are often found to exhibit, compared to human perspectives, extreme environments, such as high temperature, high salt and acidic habitats. Many archaeal species are hyperthermophiles, i.e., microorganisms that grow optimally at temperatures above 80 °C. In hyperthermophiles, as in other organisms, metabolic reactions and other essential biological processes are catalyzed by enzymes, which are optimally active where mesophilic enzymes (optimally active at 20 − 50 °C) loose their structure and function.

There appears to be no single mechanism for thermal stability. Instead, a combination of small − but relevant − structural changes, such as an increased number and optimal placement of ionic interactions and a reduction of the solvent-exposed hydrophobic surface, seem to be contributing to the enhanced thermotolerance. A better insight in what factors that make these enzymes able to retain their structure and function at high temperatures, will lead to an increased knowledge of protein stability, function and folding in general. This can in turn result in improved industrial processes and let us better understand diseases caused by incorrectly folded proteins, such as Alzheimer's, ALS and cystic fibrosis.

This licentiate thesis is mainly focused on the structure determination of Methanocaldococcus jannaschii nucleoside kinase (MjNK) by X-ray crystallography. Nucleoside kinase is a member of the ribokinase family, present in all domains of life. However, now structure is currently available for an archaeal representative of this protein family. The three-dimensional structure of MjNK will provide additional information on characteristics regarding thermal stability, enzyme mechanism and evolution of the ribokinase family. In addition, the stability of MjNK was studied by differential scanning calorimetry.

Nucleoside kinase is a homodimer with 34 kDa subunits. In the presence of ATP and Mg2+, MjNK is able to phosphorylate a wide range of nucleosides and shows the highest catalytic activity for cytidine, inosine, guanosine, and adenosine. The apparent melting temperature was 90 °C at pH 7.0. Moreover, the enzyme shows a kinetically dependent transition in 10 mM glycine pH 3.0. The three-dimensional structure of MjNK was determined by the multiple-wavelength anomalous dispersion technique using a Se-Met derivative. Additional crystal structures were determined for the apo-enzyme at 1.7 Å and MjNK in complex with an ATP-analogue and adenosine at 1.9 Å. Nucleoside kinase comprises one alpha/beta domain and a smaller lid domain. The enzyme has an overall fold homologous to the members of the ribokinase superfamily and is assumed to catalyze the phosphorylation reaction similarly to the superfamily members. MjNK shares the highest structural similarity to ribokinase from E. coli.

The structures of MjNK and ribokinase were compared regarding determinants for thermal stability. Relative to ribokinase, MjNK shows an increased charged and a decreased hydrophobic accessible surface area, a higher amount of charged residues as well as ionic networks and large aromatic clusters, characteristics that frequently are observed in enzymes from hyperthermophiles.